Articular cartilage defects are the major source of pain and functional impairment in patients with arthritis. Current treatments, whilst alleviating some of the clinical symptoms, prove insufficient to cure the underlying irreversible cartilage loss. About 43 million elderly individuals in the US currently suffer from arthritis-induced disabilities, which cause annual costs to our society in medical care and lost wages in the order of $95 billion. Stem cell transplants provide a potentially curative therapeutic option. Human induced pluripotent stem cells (hiPS cells) represent a novel cell type, which integrates advantages of human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs). hiPS cells are based on non-pluripotent cells, such as adult fibroblasts, which are reprogrammed via introduction of transcription factors that are linked to pluripotency. After a few weeks in culture, hiPS cells are virtually indistinguishable from hESCs with regards to their surface markers, morphology, proliferation, feeder dependence, gene expression and in vitro differentiation. hiPS cells have the exceptional characteristic that they are autologous and thus, able to transform into patient specific stem cells. This feature helps to avoid immune reactions and overcomes ethical concerns that are associated with hESC transplants. On the other hand, hiPS cells also overcome limitations associated with bone marrow derived hMSCs or cartilage derived chondrocytes, such as invasive harvesting procedures, variable yields, and limited cartilage regeneration potential in cells obtained from older patients. hiPS cells can be harvested as a homogeneous cell population in a consistent and reproducible manner, are easily expanded, and may be better directed to form functional 3D tissue in an in vivo environment. Thus, hiPS cells currently represent the most promising cell type for cartilage restoration. In order to regenerate hyaline cartilage in arthritic joints, the transplanted stem cells must not only survive and engraft, but also differentiate into chondrocytes. Our project endeavors to investigate molecular pathways that determine chondrogenic stem cell differentiation. We attempt to use a novel, innovative imaging approach to solve a challenging technical and conceptual problem that being our current inability to diagnose cellular differentiation processes in vivo. The goal of this study is to develop a novel, "smart" imaging technique for non-invasive in vivo visualization of the differentiation of hiPS cells into chondrocytes, based on cellular MR imaging and gene expression mediated activation of the 2-galactosidase-sensitive MR contrast agent EgadMe. The approach relies on vector-transfected hiPS cells which are marked by a cell lineage-specific expression of 2-galactosidase after chondrogenic differentiation and the galactopyranose- coated MR contrast agent EgadMe, which generates only an MR signal after cleavage by 2-galactosidase. The chondrogenic differentiation of EgadMe-labeled hiPS cells will be detected with MR imaging when the galactopyranose coat is cleaved by 2-galactosidase expression in chondrocytes, resulting in activation of the contrast agent and a positive signal on MR images. In a three step approach, we will first generate hiPS cells that express 2-galactosidase upon chondrogenic differentiation, then investigate the MR signal characteristics of EgadMe labeled hiPS cells before and after chondrogenic differentiation in vitro, and finally compare longitudinal changes in MR signal characteristics of EgadMe labeled hiPS cells before and after chondrogenic differentiation in vivo, in an established animal model of arthritis. The EgadMe-enhanced MR imaging technique may provide a novel outcome measure for stem cell differentiation, which could significantly improve monitoring of MASI and ultimately help to optimize our efforts to restore joint functions of patients with arthritis. To the best of our knowledge, the proposed approach for a non-invasive in vivo visualization of hiPS cell differentiation with an "activatable" contrast agent for MR imaging is novel and has not been described before. With the realization that novel cellular imaging techniques enable an improved in vivo characterization of physiological changes of transplanted stem cells, we predict that our MR-based imaging assay for visualizing hiPS cell differentiation will facilitate the detection and quantification of hiPS cell differentiation outcomes in vivo. Once established, this technique could be extended to characterizations of in vivo differentiation processes of any stem cell derived regenerative tissue. Potential applications comprise comparative in vivo investigations of different stem cell types (hESC, hMSC, hiPS), comparisons of the differentiation properties of autologous and allogeneic stem cells, investigations of genetically engineered stem cells, comparisons of different scaffolds and growth factors, and assessments of demographic effects on stem cell differentiation outcomes. This novel imaging technique could help to identify and monitor the most promising approaches for stem cell mediated joint restoration which could lead to the development of better matrix-associated stem cell implantation techniques, provide the long awaited functional restitution and pain relief for patients with arthritis, and eliminate direct and indirect costs associated with long term disabilities. The proposed molecular imaging technique could be used as a critical tool for preclinical assessments of stem cell-based therapies, for the design of related clinical trials, and ultimately, for the assessment and optimization of those stem cell-based therapies in clinical practice. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a magnetic resonance (MR) imaging technique that can detect the critical differentiation of human induced pluripotent stem cells (hiPS cells) into chondrocytes via activation of the novel MR contrast agent EgadMe. EgadMe provides only a signal when cleaved by the enzyme 2-galactosidase, which is produced by chondrocytes, but not undifferentiated hiPS cells. Realization of this new imaging technique could provide direct measures of in vivo stem cell differentiation outcomes which would significantly enhance our ability to identify factors that lead to cartilage regeneration and ultimately direct us to the development of successful techniques for joint restoration and functional improvement.